Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.26 (invertase)
4,927 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We fused the yeast-derived sequences encoding the invertase, acid phosphatase and alpha-factor pre- and prepro-signal peptides (SP) to the Cyamopsis tetragonoloba (guar plant) alpha-galactosidase(alpha Gal)-encoding gene and expressed these gene fusions in yeast. Whereas the amount of fusion protein produced by each of the constructs did not vary significantly, the secretion efficiency of the fusion protein that carried the SP of the prepro-alpha-factor (MF alpha 1) was consistently found to be about 10% higher than that of the other fusions (99% vs. 90%). Furthermore, when the secretion of alpha Gal was directed by the invertase (SUC2) SP, the intracellular enzyme localized to the endoplasmic reticulum (ER), whereas use of the MF alpha 1 SP caused the intracellular enzyme to be outer-chain-glycosylated and processed by the KEX2 endoproteinase, implying that it had passed the ER. These results suggest that the pro-peptide of MF alpha 1 stimulates the efflux of the heterologous protein from the ER. Null mutants of PMR1 (encoding a Ca(2+)-dependent ATPase) are known to give higher secretion efficiencies for a number of different heterologous proteins. Therefore, we also studied the secretion of alpha Gal in a pmr 1 disruption mutant. Structural analysis of the enzyme secreted by the mutant cells showed that it was completely processed by KEX2 and outer-chain-glycosylated, although the length of the outer-chain carbohydrate moiety was reduced when compared with the enzyme secreted by wild-type cells. These results contradict the hypothesis advanced by Rudolph et al. [Cell 58 (1989) 133-145] that disruption of PMR1 causes the secretory pathway to bypass the Golgi apparatus.
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PMID:Effect of a pmr 1 disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba alpha-galactosidase by Saccharomyces cerevisiae. 838 51

Expression in the yeast Saccharomyces cerevisiae of the intact nprE gene of Bacillus subtilis, which encodes the pre-pro-NprE neutral protease precursor, resulted in intracellular accumulation of unprocessed precursor without detectable secretion or processing of the expressed gene product. When sequences specifying the signal peptide of yeast invertase were fused upstream of sequences encoding the mature NprE enzyme, nprE gene products were secreted into the culture medium. The secreted protein products were, however, highly, glycosylated and biologically inactive.
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PMID:Expression of Bacillus subtilis neutral protease gene (nprE) in Saccharomyces cerevisiae. 843 52

The cDNA sequence encoding mature human C9 protein and its signal peptide was cloned into three expression vectors for expression in COS-7 (mammalian), Spodoptera frugiperda IPLB-SF-21AE (insect), and Saccharomyces cerevisiae (yeast) cells. In addition, C9 cDNA encoding only the mature protein was fused to the yeast invertase leader sequence (SUC2) and cloned for expression in yeast. Under optimal conditions COS-7 and IPLB-SF-21AE cells secreted recombinant C9 (rC9) at concentrations of about 111 and 700 ng C9/ml culture supernatant, respectively. By comparison S. cerevisiae, whether transformed with C9 cDNA containing its native or yeast invertase leader sequence, secreted only very small amounts of rC9 (5-10 ng/ml). However, upon lysis concentrations of up to 500 ng/mg dry wt were found in yeast cells transformed with C9 cDNA. SDS-PAGE followed by Western blot analysis revealed COS-7 cell and S. cerevisiae expressed rC9 to have a MW similar to that of native C9 purified from human serum, while rC9 from IPLB-SF-21AE cells was about 4 kDa smaller. No hemolytic activity of S. cerevisiae secreted rC9 could be detected and the specific hemolytic activity of S. cerevisiae intracellular rC9 was also very low. However, the specific hemolytic activities of COS-7 and IPLB-SF-21AE secreted rC9 were indistinguishable from that of purified native human C9. Thus, for future studies on the structure and function of C9 where the production of large quantities of mutant protein would be desirable, the baculovirus-insect cell expression system appears to offer considerable advantages.
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PMID:The expression of hemolytically active human complement protein C9 in mammalian, insect, and yeast cells. 847 47

Gene fusions have provided a strategy for determining the topology of polytopic membrane proteins in Escherichia coli. To evaluate whether this highly effective approach is applicable to heterologously expressed eukaryotic integral membrane proteins, we have carried out a comparative topological study of the eukaryotic membrane protein Ste6 both in bacteria and in yeast. Ste6, is an ATP binding cassette (ABC) protein, essential for export of the a-factor mating pheromone in Saccharomyces cerevisiae. The topogenic reporters, invertase in S. cerevisiae and alkaline phosphatase in E. coli, were fused to Ste6 at identical sites and the fusions were expressed in yeast and bacteria, respectively. The results obtained in both systems are similar, although more definitive in E. coli, and support the predicted six-transmembrane spans organization of the N-terminal half of Ste6. Thus, the topological determinants for membrane insertion of polytopic proteins in prokaryotic and in eukaryotic systems appear to be highly similar. In this study we also demonstrate that Ste6 does not contain a cleaved signal sequence.
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PMID:Comparative topology studies in Saccharomyces cerevisiae and in Escherichia coli. The N-terminal half of the yeast ABC protein Ste6. 866 64

A number of studies have introduced mutations into the yeast invertase signal peptide, using it as a model system to elucidate features for targeting, translocation and intracellular transport. Using molecular modelling of the invertase signal peptide we have analysed the hydrophobicity potential and the change in dielectric constant of the energy transfer, when the molecule moves from a hydrophobic to a hydrophilic phase at the simulated hydrophobic-hydrophilic interface. This modelling has been carried out on wild type and mutant invertase signal peptides of altered function, previously reported in the literature. While the predicted angle of insertion correlates with the measured extent of invertase secretion, with an optimum angle of 45 degrees, mutations that change the angle of orientation reduce the extent of invertase secretion. We have applied these same molecular modelling principles to the naturally occurring variants of the human apolipo-protein B (apoB) signal peptide, that confer a secretion defective phenotype when fused to yeast invertase and expressed in yeast. Our modelling thus identifies a strong correlation between the predicted angle of insertion of the signal peptide into the membrane and its ability to direct secretion.
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PMID:Prediction of signal peptide functional properties: a study of the orientation and angle of insertion of yeast invertase mutants and human apolipoprotein B signal peptide variants. 873 6

When the extracellular domain of rat low-affinity nerve growth factor receptor (NGFRe) was synthesized in Saccharomyces cerevisiae with the signal peptide of invertase, NGFRe was translocated to the endoplasmic reticulum (ER) and retained there. However, when NGFRe was fused to the C-terminus of the hsp150 delta-carrier, the hsp150 delta-NGFRe fusion protein was efficiently secreted to the growth medium with no apparent retention in the ER. The NGFRe portion was disulphide-bonded and its single N-glycosylation site was occupied. The hsp150 delta-carrier is an N-terminal signal peptide-containing fragment of a yeast secretory glycoprotein. Hsp150 delta-NGFRe, harvested from the culture medium, inhibited the cross-linking of [125I]NGF to authentic NGFR on the surface of human melanoma cells. Moreover, [125I]NGF could be chemically cross-linked to secretory hsp150 delta-NGFRe, suggesting that the NGFRe portion had adopted a ligand-binding conformation. However, inhibition of the cross-linking by unlabelled NGF was less effective than in the case of the authentic receptor. The hsp150 delta-carrier may have potential in the production of mammalian proteins, which require elaborate folding and disulphide formation in the ER.
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PMID:The hsp150 delta-carrier confers secretion competence to the rat nerve growth factor receptor ectodomain in Saccharomyces cerevisiae. 874 Apr 19

The 5' cap structure of eukaryotic mRNAs is believed to play a role in a number of cellular processes, including pre-mRNA splicing, nuclear export and translation. An essential cap-binding protein that is likely to mediate the participation of the cap in at least one of these processes is the eukaryotic translation initiation factor eIF4E. This protein is thought to facilitate the initial ribosomal interaction with the 5' end of the mRNA, involving the binding of eIF4E to the cap in the cytoplasm. Yet the subcellular distribution and mechanism of targeting of eIF4E has been an unresolved issue. We have therefore examined whether eIF4E in the yeast Saccharomyces cerevisiae is directed to the nucleus by virtue of a nuclear localization sequence (NLS) in its amino acid sequence. eIF4E was fused with the "marker proteins' yeast invertase and jellyfish green fluorescent protein. The distribution of these fusions could be followed using immunofluorescence and confocal microscopy of protoplasts and whole cells. These and other fusions were used to show that while yeast eIF4E does not possess an efficiently functioning NLS, it can be transported into the nucleus if provided with a known active NLS. However, an NLS-eIF4E fusion of this type cannot be stably supported by the cell, most likely because of its inhibitory effects when present in large quantities in the nucleus, whereas an NLS fusion with a mutant form of eIF4E that has reduced cap-affinity is tolerated.
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PMID:Intracellular targeting and mRNA interactions of the eukaryotic translation initiation factor eIF4E in the yeast Saccharomyces cerevisiae. 876 32

An efficient expression/purification procedure has been developed which allows the production of pure, biologically active recombinant leech-derived tryptase inhibitor (rLDTI), originally found in the leech Hirudo medicinalis. The gene for LDTI was generated synthetically from three overlapping oligonucleotides by PCR synthesis. LDTI was expressed in the yeast Saccharomyces cerevisiae under the control of the copper-inducible CUP1 promoter and fused to the invertase signal sequence (SUC2). The entire expression cassette was inserted into the yeast high-copy vector pDP34. Appropriate host strains transformed with the expression plasmid secreted rLDTI into the medium upon copper addition. Proteinchemical analysis of the secreted rLDTI revealed exclusively inhibitor with the correct N-terminal sequence. Up to 60% of the rLDTI, however, appeared to be modified by glycosylation and the unglycosylated material showed heterogeneity at the C-terminus. Besides full-length rLDTI, truncated rLDTI species lacking either the terminal Asn46 or in addition the penultimate Leu45 were isolated. The C-terminally truncated variants were eliminated using a S. cerevisiae host strain disrupted in the structural genes of carboxypeptidases yscY and ysca, thus identifying these proteases as being responsible for the degradation of rLDTI. Mature rLDTI was purified in high yields from the culture supernatant of the carboxypeptidase-deficient yeast strain by cation-exchange chromatography and reverse-phase HPLC. The recombinant protein is at least 98% pure, based on HPLC and capillary electrophoresis, and is fully biologically active. Structural identity with the authentic leech protein was confirmed by sequence analysis and molecular-mass determination. The purified protein was tested for its ability to inhibit tryptase and trypsin in vitro and to interfere with the tryptase-induced proliferation of human fibroblasts and keratinocytes. Recombinant LDTI appears to be as potent as the authentic leech protein, exhibiting Ki-values of approximately 1.5 nM and approximately 1.6 nM against human tryptase and bovine trypsin, respectively. The tryptase-induced proliferation of human fibroblasts and keratinocytes was inhibited with half-maximum values of approximately 0.1 nM and approximately 1 nM, respectively. The availability of the recombinant material will allow evaluation of the concept of tryptase inhibition in various disease models and to test the therapeutic potential of LDTI in mast-cell-related disorders.
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PMID:Purification, characterization and biological evaluation of recombinant leech-derived tryptase inhibitor (rLDTI) expressed at high level in the yeast Saccharomyces cerevisiae. 891 64

Genetic constructs in which different N- and C-terminal segments of Brazil nut (Bertholletia excelsa H.B.K.) 2S albumin were fused to secretory yeast invertase were transformed into tobacco (Nicotiana tabacum) plants to investigate the vacuolar targeting signal of the 2S albumin. None of the N-terminal segments, including the complete precursor containing all propeptides, was able to direct the invertase to the vacuoles. However, a short C-terminal segment comprising the last 20 amino acids of the precursor was sufficient for efficient targeting of yeast invertase to the vacuoles of the transformed tobacco plants. Further analyses showed that peptides of 16 and 13 amino acids of the C-terminal segment were still sufficient, although they had slightly lower efficiency. When segments of 9 amino acids or shorter were analyzed, a decrease to approximately 30% was observed. These segments included the C-terminal propeptide of four amino acids (Ile-Ala-Gly-Phe). When the 2S albumin was expressed in tobacco, it was also localized to the vacuoles of mesophyll cells. If the C-terminal propeptide was deleted from the 2S albumin precursor, all of this truncated 2S albumin was secreted from the tobacco cells. These results indicate that the C-terminal propeptide is necessary but not sufficient for vacuolar targeting. In addition, an adjacent segment of at least 12 amino acids of the mature protein is needed to form the complete signal for efficient targeting.
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PMID:The vacuolar targeting signal of the 2S albumin from Brazil nut resides at the C terminus and involves the C-terminal propeptide as an essential element. 893 6

PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single-polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal.
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PMID:Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe. 920 31


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